A Method for Cuticle Growth using Granulocyte-Colony Stimulating Factor

ABSTRACT

The present invention provides a method for re-growing hair in patients with androgenic alopecia by administering effective consecutive courses of Granulocyte-Colony Stimulating Factor or derivatives. After the state of re-growth is obtained, the hair growth is maintained by administering periodic courses of Granulocyte-Colony Stimulating Factor or derivatives. The invention further provides a method for increasing cuticle growth and density using a similar administration of effective consecutive courses of Granulocyte-Colony Stimulating Factor or derivatives. The increased cuticle growth and density is maintained by administering periodic courses of Granulocyte-Colony Stimulating Factor or derivatives.

FIELD OF INVENTION

A method for reversing hair loss and the loss of cuticle growth anddensity by administering effective courses of Granulocyte-ColonyStimulating Factor.

BACKGROUND OF THE INVENTION

Parkinson's disease is an example of a progressive neurodegenerativedisease. It is usually diagnosed in adulthood, usually when the patientis about 55 years of age, and is characterized by tremors, rigidity, andbradykinesia. It is well understood that many of the abnormalities foundin these patients are due to the loss of dopamine (DA) neurons in thesubstantia nigra and the depletion of striatal dopamine levels. Morerecently, there has been a re-evaluation of the signs and symptoms foundin Parkinson's disease patients that has led to the conclusion thatParkinson's disease is a systemic disease with involvement of peripheralnervous tissue. For example, the loss of the sense of smell has beenfound to be an early sign of Parkinson's disease reported in mostpatients. Often, the earliest sign of Parkinson's disease in patients isthe loss of the sense of smell so that this symptom can be used as adiagnostic in patients with early disease. This symptom of disease isprobably due to loss of neurons in the olfactory bulb of the brain.Also, orthostatic hypotension has been shown to be one of the morecommon signs of Parkinson's disease, probably due to a direct effect onthe peripheral nervous system (PNS) of the host. Clinical manifestationsof Parkinson's disease are not apparent until over 80% of the central orperipheral neurons have degenerated. Most new Parkinson's diseasepatients are started on dopamine (DA) agonists when first diagnosed, butusually progress to L-DOPA (L-dopamine), the precursor of dopamine intissue. As a therapeutic, L-DOPA is used to relieve Parkinsonian motorsigns, but has very little effect on PNS signs and symptoms of disease.In fact, its long-term use is usually associated with diminishedefficacy and increasingly bothersome side effects. Other examples ofchronic degenerative neurological disorders that might be treated in asimilar manner include macular degeneration, urinary incontinence,Alzheimer's disease, Multiple Sclerosis, and short term memorydeficiency. These disorders have in common three characteristics; theyare usually diagnosed late in life, there is evidence of familial, butnot Mendelian genetic inheritance of each disease, and patients willhave had a period of time as adults before diagnosis of disease when thehost appears to function normally without evidence of signs or symptomsof the chronic disease.

Over the past few years, tremendous strides have been made inunderstanding the crucial role that stem cells play in embryogenesis andorganogenesis. Much of the accumulated data indicates that stem cellsplay an important role in the development and maturation of mammals. Itis well known that each organ of the adult body contains progenitor stemcells that can respond to signals from other cells or injured tissue andmigrate to the site of injury to help restore the tissue to normalhealth. In animal models, the “self-repair” system clearly responds toacute injury of tissues in the body and recruits stem cells from thebone marrow and other parts of the body to help with the repairs. Stemcells are also required to sustain those functional cell populationsthat turn over rapidly in the body such as skin and the variouslymphocyte populations. The “self-repair” system is only now beingdemonstrated in human studies.

With the beginning of an understanding of the “self-repair” system andits probable role in response to tissue injury, researchers haveproposed that delivering somatic progenitor stem cells directly to thesite of damage might augment the hosts own “self-repair” system and morequickly and completely repair the damage to the host tissue. Forexample, neural stem cell lines have been successfully used to treatspinal cord injuries mice and rats. However, this “self-repair” responseappears to be determined by a complex interaction between cells andprotein mediators produced by host and donor cells. Understanding thefactors involved and their roles in this response and the differentiatedstate of the cells involved will be crucial to devising methods forcontrolling and utilizing this system for providing new therapies forchronic diseases. Studies are ongoing in many laboratories to furtherdefine the factors and cells involved. Most recently, continuousinfusion of Glial-Derived Neurotrophic Factor (GDNF), a stem cell growthfactor, directly into the brain has been claimed to have benefit forpatients with Parkinson's disease.

Over the past several years, significant interest has developed in usingmobilized peripheral blood progenitor cells for allogeneic hematopoieticreconstitution. Treatment of stein cell donors with a five-day course ofGranulocyte-Colony Stimulating Factor or its pegalated derivative causesthe release of stem cells from the bone marrow into the circulatingblood and greatly increases the number of hematopoietic and other steincells that could be harvested from the donor. This procedure requiresGranulocyte-Colony Stimulating Factor be administered to otherwisenormal donors in order to release stem cells into the peripheral bloodwhere they can be collected by leukophoresis and prepared fortransplantation. Many studies have reported the use ofGranulocyte-Colony Stimulating Factor in normal volunteers and normaldonors, usually at a dose of 5 to 10 micrograms per kg per day for 4 to8 days. The most common side effect was bone pain. While the toxicitieswere frequent, the severity was generally mild and very few normaldonors had to discontinue Granulocyte-Colony Stimulating Factor becauseof the side effects. Persons treated with Granulocyte-Colony StimulatingFactor were found to have a surge in peripheral blood stem cells 4 to 7days after initial treatment. The use of Granulocyte-Colony StimulatingFactor for mobilizing peripheral blood stem cells is widespread andappears to be safe and to be capable of generating the stem cells neededfor allogenic or autologous transplantation.

Although the above studies suggest Granulocyte-Colony Stimulating Factoris capable of mobilizing peripheral hematopoietic stem cells, it is notknown if Granulocyte-Colony Stimulating Factor could induce therecruitment of either local or migration of peripheral stem cells toinjured neural tissue, differentiate and restore neural functionrequired for the slowly developing lesions found in most chronicdiseases. Further, to our knowledge a “self-repair” system has not beendescribed in human studies.

Diabetes is a major public health problem in the United States affecting16 million people and accounts for one sixth of all health relatedexpenditures. There are two types; Type 1 (insulin dependent diabetes)and Type 2 (noninsulin-dependent diabetes). Type 1 is characterized bybeta cell loss and absolute insulin deficiency. Of the patients withdiabetes today, approximately 90 to 95% of the inflicted are Type 2diabetics. It is generally characterized by elevated fasting bloodglucose and lack of sensitivity to insulin and impaired insulinsecretion. The prevalence of Type 2 diabetes is about 7 percent forpersons between 45 to 64 years of age. The microvascular andmacrovascular complications of Type 2 diabetes causes significantmorbidity and mortality in affected individuals. Diabetic retinopathy,neuropathy, and nephropathy are major causes of functional limitationsand disability in this patient population. In the event that diet andexercise are not sufficient to control blood glucose, diabetics may betreated with one, and typically two, of several oral drugs able to lowerblood glucose levels which include sulfonylureas, metformin,alpha-glucosidase, troglitazone, and repaglinide. These agents act onone of four mechanisms that alter renal function, liver metabolism,insulin secretion or breakdown of complex carbohydrates. If these drugsare insufficient, insulin treatment may be prescribed alone or togetherwith these oral agents.

Improved glycemic control reduces the risk of microvascularcomplications in Type 2 diabetes. Despite this evidence, patients withType 2 diabetes frequently do not maintain adequate glycemic control.However, the health outcomes of patients with Type 2 diabetes who aretreated with insulin to control glycemia do much better than those thatdo not. Patients are encouraged to use intensive insulin treatmentprotocols to better control blood sugar but analysis of their outcomesindicate that it did not affect the quality of life of patients in theintensive insulin treatment nor did it have a significant protectiveeffect against cardiovascular diseases. There is evidence that tightglycemic control will decrease the incidence of microvascularcomplications so patients should be encouraged to use insulin and oralhypoglycemic agents. However, it is difficult to make a convincingargument to patients that do not currently have severe symptoms ofdisease associated with their elevated blood sugar levels. There are noother forms of medical treatment to lower blood glucose to an acceptablerange. The ideal drug for these patients is one where a single drug canbe taken periodically that is able to control blood glucose levels overthe course of a month or longer with reduced side affects.

It is unknown whether the chronic progressive neurodegenerative andnon-neurodegenerative disorders could be treated effectively bymobilizing the “self-repair” mechanism of the host or even if that“self-repair” mechanism could be detected in patients with thesedisorders. Furthermore, Parkinson's disease in humans is a systemicdisease with symptoms that indicate the PNS is an important targettissue of this disease. Surprisingly, this invention directlydemonstrated for the first time in humans the potential action ofGranulocyte-Colony Stimulating Factors, known to mobilize stem cellsinto the peripheral blood as well as to cause them to differentiate, toalso be a therapy to reverse symptoms of an adult onsetneurodegenerative disorder such as Parkinson's disease. In addition, itwas even more surprising that Type 2 diabetes was found to beeffectively controlled by periodically administering ofGranulocyte-Colony Stimulating Factors offering a new approach and arevolutionary treatment for this disease. In both cases of adult onsetdisease, the patient receiving the drug was provided a long termreversal of all disease symptoms and this allowed him to live a morenormal lifestyle. Also, the extended period of time between courses ofGCSF might also result in a lower incidence of side effects from drugtherapy.

SUMMARY OF THE INVENTION

The present invention is directed to provide a method of treatingsymptoms associated with a neurodegenerative disease in a human byadministering an effective dose of Granulocyte-Colony Stimulating Factoror its derivatives or combinations thereof. More preferably, theinvention is directed to provide a method for the reversal of symptomsassociated with Parkinson's Disease in a human through periodicallyadministering Granulocyte-Colony Stimulating Factor, or its derivatives,or combinations thereof.

In another aspect, the invention herein is directed to provide a methodof treating symptoms associated with diabetes in humans by administeringan effective dose of Granulocyte-Colony Stimulating Factor or itsderivatives, or combinations thereof. More preferably, the invention isdirected to provide a method for the extended reduction of blood glucoselevels associated with Type 2 diabetes in a human through administeringof Granulocyte-Colony Stimulating Factor, or its derivatives, orcombinations thereof.

In the first aspect of the invention, one or more symptoms of an adultonset neurodegenerative disease are treated by administering to a humanan effective dose of Granulocyte-Colony Stimulating Factor, or itsderivatives, or combinations thereof. Adult onset neurodegenerativediseases include but are not limited to Parkinson's Disease, maculardegeneration, urinary incontinence, age related short term memory loss,and multiple sclerosis. More preferably, one or more symptoms of adultonset Parkinson's disease are reversed by administering an effectivedose of Granulocyte-Colony Stimulating Factor or its derivatives orcombinations thereof.

In the second aspect of the invention, an effective dose ofGranulocyte-Colony Stimulating Factor, or its derivatives, orcombinations thereof is administered to a human with an adult onsetneurodegenerative disease by subcutaneous injection, transdermal patch,intravenously, orally or other means. A typical period for administeringGranulocyte-Colony Stimulating Factors is from about 1 to 8 days,preferably ranging 3 to 6 days.

In a third aspect of the invention an effective dose ofGranulocyte-Colony Stimulating Factor, or its derivatives, orcombinations thereof are administered daily to a human for treatment ofan adult onset neurodegenerative disease ranging from 0.1 micrograms to20000 micrograms per kg body weight per day, preferably between 1 to 20micrograms per kg body weight per day.

In a fourth aspect of the invention an effective dose ofGranulocyte-Colony Stimulating Factor or its derivatives, or itsderivatives, or combinations thereof is administered to a human fortreatment of an adult onset neurodegenerative disease for about 1 to 8days and repeated about every 2 to 18 weeks, and more preferably every 4to 10 weeks.

In a fifth aspect of the invention an effective dose ofGranulocyte-Colony Stimulating Factor or its derivatives, orcombinations thereof is administered into a human for the reversal ofone or more symptoms associated with Parkinson's Disease. Symptoms ofParkinson's Disease may be either central nervous system or peripheralnervous system derived and, include but are not limited to, orthostatichypotension, resting tremor, rigidity, postural instability,micrographia, urinary and gastrointestinal incontinence and lack ofsense of smell.

In a sixth aspect of the invention an effective dose ofGranulocyte-Colony Stimulating Factor or its derivatives, orcombinations thereof is administered into a human with an adult onsetneurodegenerative disease or condition in combination with stem cells,selected from a group consisting of harvested adult progenitor stemcells or stem cell lines. In a related aspect, harvested adultprogenitor stem cells or stem cell lines are pre-treated withGranulocyte-Colony Stimulating Factor or its derivatives or combinationsthereof before injection.

In a seventh aspect of the invention, the level of blood glucose in anadult onset Type 2 diabetic is reduced by treatment with an effectivedose of Granulocyte-Colony Stimulating Factor or its derivatives, or itsderivatives, or combinations thereof. Preferably the blood glucoselevels after treatment remain reduced by 2 days longer, more preferably1 week or longer and most preferably 4 weeks or longer.

In the eighth aspect of the invention, an effective dose ofGranulocyte-Colony Stimulating Factor or its derivatives or combinationsthereof is administered into a human for treatment of diabetes, andpreferably Type 2 diabetes, by subcutaneous injection, transdermalpatch, intravenously, orally or other means. A typical period foradministering is from about 1 to 8 days, more preferably ranging 3 to 6days.

In the ninth aspect of the invention an effective dose ofGranulocyte-Colony Stimulating Factor, or its derivatives, orcombinations thereof administered into a human for treatment of diabetesranging from 0.1 micrograms to 20000 micrograms per kg body weight perday, preferably between 1 micrograms to 20 micrograms per kg body weightper day.

In the tenth aspect of the invention an effective dose ofGranulocyte-Colony Stimulating Factor or its derivatives, orcombinations thereof is administered for treatment of Type 2 diabetesfor about 1 to 8 days and the treatment is repeated about every 2 to 18weeks, and preferably every 4 to 10 weeks. Preferably,Granulocyte-Colony Stimulating Factors is administered at least one weekbefore the blood glucose levels rise to the levels prior to treatment.

In an eleventh aspect of the invention, Granulocyte-Colony StimulatingFactor or its derivatives, or combinations thereof is effective inimproving other brain associated neural diseases that include but arenot limited age related memory impairment, Schizophrenia, andAlzheimer's.

In the twelfth aspect of the invention, an effective dose ofGranulocyte-Colony Stimulating Factor, or its derivatives, orcombinations thereof for accelerating hair re-growth or cuticle growthand density when administered for about 3 to 6 days and the treatment isrepeated about every 3 to 8 days for two or more consecutive courses,preferably 3 to 6 courses. An effective dose of Granulocyte-ColonyStimulating Factor, or its derivatives, or combinations thereof areadministered daily to a human for treatment for hair re-growth orcuticle growth ranging from 0.1 micrograms to 20000 micrograms per kgbody weight per day, preferably between 0.1 to 10 micrograms per kg bodyweight per day.

In the thirteenth aspect of the invention, an effective dose ofGranulocyte-Colony Stimulating Factor, or its derivatives, orcombinations thereof for maintaining hair growth or cuticle growth anddensity when administered for about 3 to 6 days and the treatment isrepeated about every 2 to 18 weeks, and preferably every 6 to 12 weeks.An effective dose of Granulocyte-Colony Stimulating Factor, or itsderivatives, or combinations thereof are administered daily to a humanfor hair treatment from 0.1 micrograms to 20000 micrograms per kg bodyweight per day, preferably between 0.1 to 10 micrograms per kg bodyweight per day.

In the fourteenth aspect of the invention, an effective dose ofGranulocyte-Colony Stimulating Factor or its derivatives, orcombinations thereof is administered into a human to restore or maintainhair growth or cuticle growth in combination with adult stem cells,selected from a group consisting of or stem cell lines or harvestedadult donor stem cells or progenitor stem cells from an individualadministered with Granulocyte-Colony Stimulating Factor or itsderivatives. Other compounds that increase the mobilization of stemcells when used with Granulocyte-Colony Stimulating Factor or itsderivatives may optimize production of donor adult stem cells. Adultstem cells obtained from self from 16 to 35 years of age and morepreferably from self from 20 to 25 years of age. In a related aspect,adult stem cells, progenitor stem cells or stem cell lines arepre-treated with Granulocyte-Colony Stimulating Factor or itsderivatives or combinations thereof before injection.

“Granulocyte-Colony Stimulating Factors” means Granulocyte-ColonyStimulating Factor or its derivatives, or other biologically orchemically derived compounds or factors that are functional equivalent.

“Maintaining” means slowing, interrupting, arresting or stopping theprogression of the disease or condition,

“Reversing” means the improvement of one or more symptoms from thediseased state or condition rather than maintaining the current state ofdisease.

“Re-growth” means the growth of new hair in the scalp and other areasdue to conditions of hair loss, preferably adult onset androgenicalopecia, rather than increased hair growth.

“Treat” means maintaining the state of the disease or condition or thereversal of a disease symptom or condition,

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the long term reversal of Parkinson's Diseasesymptoms using the United Parkinson's Disease Scale to evaluate thecondition of the patient administered with Granulocyte-ColonyStimulating Factor over two and one-half years.

FIG. 2 demonstrates the reduction of blood glucose levels below thepreferred normal limit after Granulocyte-Colony Stimulating Factor wasadministered to Type 2 diabetic patient.

FIG. 3 shows the average time course for blood glucose levels of sixtreatments with GCSF.

FIG. 4 shows a GCSF treated identical twin and untreated identical twinwith different hair losses.

DETAILED DESCRIPTION OF INVENTION

The present invention described herein was aimed at performingtreatments using Granulocyte-Colony Stimulating Factors administered topatients with neurodegenerative diseases, more preferably Parkinson'sdisease, to determine whether Granulocyte-Colony Stimulating Factorswere able to maintain the state of the diseases, and preferably reversethe symptoms, either partially or completely. Unexpectedly, it was foundthat the treatment of the neurodegenerative disease, Parkinson'sDisease, with Granulocyte-Colony Stimulating Factors reversed symptomsof both the central nervous system (CNS) and peripheral nervous system(PNS). In addition, it was found that Granulocyte-Colony StimulatingFactors delivered in an effective dose was able to reduce blood sugarlevels of patients with adult onset Type 2 diabetes for a significantperiod of time.

The nucleic acid sequence and encoded amino acid sequence ofGranulocyte-Colony Stimulating Factor (GCSF), also referred to as humanpluripotent granulocyte colony-stimulating factor, as well as chemicallysynthesized polypeptides sharing its biochemical and immunologicalproperties has been previously disclosed (U.S. Pat. Nos. 6,379,661;6,004,548; 6,830,705; 5,676,941, 6,027,720; 5,994,518; 5,795,968;5,214,132; 5,218,092; 6,261,550; 4,810,643; 4,810,321). Other examplesof Granulocyte-Colony Stimulating Factor include analogs which retainedtheir three-dimensional structures and hybrid molecules maintainingtheir biological and structural integrity were described by Osslund(U.S. Pat. No. 6,261,550). Examples of functional GCSF variants includeany proteins, peptides or fragments thereof that are at least 70%,preferably 80% and most preferably at least 90% identity to full-lengthhuman GCSF amino acid sequence or its nucleotide sequence. Modificationsof GCSF to improve functionality or resident serum clearance include butare not limited to polyethyleneglycol and polyethyleneglycol derivativesthereof, glycosylated forms (Lenogastrim™) (WO 00/44785), norleucineanalogs (U.S. Pat. No. 5,599,690), addition of amino acids at eitherterminus to improve folding, stability or targeting, and fusionproteins, such as GCSF and albumin fusion protein (Albugranin™) (U.S.Pat. No. 6,261,250). An increase in biological or functional activityover the native peptide may reduce the amount of dose and/or the timeperiod required for treatment. Any chemical or biological entity thatfunctions similar to GCSF can also be employed. GCSF, or the drug nameFilgrastim, is currently being sold as Neupogen® and its polyethyleneglycol modified or pegulated form, with the drug name Pegfilgrastim,sold as Neulasta™.

Examples of closely related functional forms includeGranulocyte-Macrophage Colony Stimulating Factor (GMCSF) whose codingDNA sequence and protein including amino acid sequence are known as wellas various methods employed to produce recombinant proteins (U.S. Pat.No. 5,641,663). Examples of functional GMCSF variants include anyproteins, peptides or fragments thereof that are at least 70%,preferably 80% and most preferably at least 90% identity to full-lengthhuman GMCSF amino acid sequence or its nucleotide sequence.Modifications of GCSF to improve functionality or resident serumclearance include but are not limited to polyethyleneglycol andpolyethyleneglycol derivatives thereof, glycosylated forms, norleucineanalogs, addition of amino acids at either terminus to improve folding,stability or targeting, and fusion proteins, such as GCSF and albuminfusion protein (Albugranin™). An increase in biological or functionalactivity over the native peptide may reduce the amount of dose and/orthe time period required for treatment. Any chemical or biologicalentity that functions similar to GMCSF can also be employed. Examples ofGMCSF, or the drug name Sargramostim, which are currently being sold,include Leukine® or Leucomax® and Leucotropin®.

In one embodiment oral dosages, and methods thereof, ofGranulocyte-Colony Stimulating Factor have been described by Nomura andKazutoshi (U.S. Pat. No. 5,597,562) that allow for dosage reductions,facilitate dose control, and increase the practical usefulness of thebioactive proteins. In addition, Brimelow and Nanette (U.S. Pat. No.6,497,689) have described preferred pH ranges comprising sulfate ionsfor stabilizations.

Granulocyte-Colony Stimulating Factors has been suggested to act as aneuroprotective agent in vitro and may be used for the potentialtreatment of diseases that result from oxidative stress or apoptosissuch as in cerebral ischemia and traumatic brain injury (U.S. Pub. No.2004/0141946). The work was focused on an in vitro model using STATproteins and GCSF receptor or rat model for cerebral ischemia. It wassuggested that GCSF may be used to “treat” broadly ischemic or hypoxicrelated diseases as well as neurological, psychiatric andneurodegenerative diseases as neuroprotective agent acting to slow,interrupt, arrest or stop the progression of the disease. However thework fails to provide insights on how treatment would work in humans asno human study was performed.

Type 2 diabetes is an example of a non-neurological disorder thatappears to be due to the lack of insulin sensitivity of the target cellsor insufficient levels of insulin in response to blood glucose. Althoughit is unknown what causes Type 2 diabetes, it is clear that the diseaseis usually first diagnosed as an adult and is usually progressive (interms of the need for therapy to control blood sugar). Type 2 diabeticswill have had a relatively long period of time with normal blood sugarbefore the fasting blood sugar levels begin to rise and the disease canbe diagnosed. Other non-neurological disorders include osteoarthritisand benign prostate hypertrophy.

In view of ongoing research work on stem cells, the use ofGranulocyte-Colony Stimulating Factors are likely to enhance or berequired in the treatment of neurodegenerative diseases by stem celltherapy. For example, a method for the differentiation of stein cells inculture using Granulocyte-Colony Stimulating Factor and other factors,including lipopolysaccharides, to obtain immune system suppressor cellsand immune systems stimulator cells was described by Ogle et al (U.S.Pat. No. 6,165,785). Using a similar approach, a method is useful indifferentiating stem cells that are destined to become replacements fordamaged cells in neurodegenerative disease and diabetes. Uponpretreatment of stem cells with Granulocyte-Colony Stimulating Factors,injected stimulated stem cells into patients with adult onset Type 2diabetes and adult onset neurodegenerative diseases, such as Parkinson'sDisease, are able to initiate repair or to enhance the effect overGranulocyte-Colony Stimulating Factors alone. The patient provided thesestem cells is also administered Granulocyte-Colony Stimulating Factorsfor further benefit. Alternatively, patient with the adult onsetdiseases is injected with stem cells that are not pretreated withGranulocyte-Colony Stimulating Factors but subsequent treatment andprovided with an effective dose of Granulocyte-Colony StimulatingFactors to enhance response.

In cases where the patient has a low or depleted level of stein cellsdue to age, being immunocompromised due to diseased, sickened state orlike condition, the preferred treatment of patients requiringsupplemental doses stem cells from those donors are those individualsthat are histocompatibility similar or that match having nearlyidentical major histocompatibility complex (MHC) such an close relativeor preferably a twin. A preferred source of stem cells is to administera dose of the Granulocyte-Colony Stimulating Factors to the patient orclose relative and collect the stem cells by methods well known in theart. The more preferred source of stem cells would be stem cellscollected from the individual when the individual is between 16 to 35years of age and most preferred when the individual is between 18 to 30years of age. Once the stem cells are collected, the stem cells may besubjected to additional rounds of Granulocyte-Colony Stimulating Factorsin vitro to increase the numbers of stein cells prior to treatment ofthe patient. In other embodiments, the multiplied stem cells aresubjected to preservation methods, freezing at low temperatures andstorage at less than −50° C. to cryopreserve the cells until needed.

To optimize the production of stem cells in an individual,Granulocyte-Colony Stimulating Factors administered in conjunction withagonist and antagonist of ligand stromal derived factor-1 (SDF-1) toreduce expression of chemokine receptor CXDR4. Antagonist such asAMD3100 block the binding of SDF-1 and is known to cause rapidmobilization of CD34+ cells. With Granulocyte-Colony StimulatingFactors, AMD3100 (brand name Mozobil or Plerixafor) is effective inincreasing the yield of CD334+ cells (J. Clin. Oncology, 2004,1095-1102). Alternatively, an agonist peptide, CTCE-0021, may be morestable than natural SDF-1, which is rapidly cleaved by serum enzymes,has been reported to be an effective mobilizing agent. Other methodssuggested to increase SDF-1 degradation are protease inhibitors such asMMP-9.

During the present invention, it was found that GCSF and likely relatedcompounds are biological factors capable of mobilizing stem cells in thebody and repairing or restoring specific functions of tissues. For anygiven cellular function, stem cells provide progenitor cells required bythe body to restore specific functions of those tissues as evidenced bythe effects on the more complex condition of Type 2 diabetes describedherein. GCSF can cause the replication of those cells in response tospecific mediators.

In another condition having a similar age related onset to Type 2diabetes is age related androgenetic alopecia, referred to as malepattern baldness, which is the main cause of hair thinning or hair lossin adults. It is know that men with androgenic alopecia have lowerlevels of testosterone and increased amount of sex hormone-bindingglobulin that binds to testoterone. Interestingly, sex hormone-bindingglobulin is down regulated by insulin. Common but less than desirabletreatments include minoxidil applied to the scalp to stimulate hairfollicles and finasteride as an oral administration to slow hair lossbut can have significant side effects. Finasteride acts to inhibit typeII 5 alpha-reductase and subsequently inhibits conversion to DHT. Otherroutine treatments include hair transplants using plugs of hair fromother areas of the scalp where hair continued to grow.

Recent investigations on improved treatments for hair loss have focusedon those stem cells that are ultimately responsible for continued growthof hair. The growth of hair is not synchronized and grows in cycles inhumans. It has three distinct phases of the hair growth cycle: anagen,catagen and telogen. The last phase, telogen, the keratinization iscompleted and the keratinized hair falls out. To replace the lost hairfollicle, a new matrix is gradually formed from progenitor stem cellsthat are gathered in the basal layer in the outer root sheath bulge ofthe hair follicle. A new hair starts to grow from the matrix and thefollicle returns to anagen, the hair growth phase.

Matrix cells derived from stem cells are under the influence ofsubstances that control their growth and differentiation produced bycells of the dermal papilla. A large number of these factors arecytokines that influence or inhibit the proliferation of hair matrixcells and hair growth. Two factors believed to impact hair growth arekeratinocyte growth factor (KGF) and insulin-like growth factor I(IGF-I) in animals.

Interestingly, nails are hardened epidermal variants of hair that growcontinuously from the nail organ. The nail organ is an epidermalinvagination on the dorsal side of the digit tips that is an equivalentof a large hair follicle. At the proximal end of the nail lies a matrix,a mass of stem cells that is equivalent to the pre-matrix cells in thehair follicle. The matrix is the immediate source of the cells thatforms the nail. Unlike the loss of hair, typically nails becomesignificantly weaker in aging as well as in response to fingernailcosmetics resulting in more brittle and flexible nails.

It has been unclear whether the loss of hair in humans, or androgenicalopecia, is caused by the inability of stem cells to mature into matrixcells that produce hair or due to a loss of stem cells or asubpopulation of stem cells that differentiate into matrix cellproducing hair or a combination of both. However, a recent studysuggested that balding might arise from stem cell activation rather areduction in the number of stem cells in follicles present (J ClinInvest 2011, 121, 613). Further, they found that the same number of stemcells in the balding area, however the number of progenitor stem cellswas markedly depleted in the scalp.

Other current approaches are exploring stem cell therapy for hair losstreatment. There has been considerable interest by numerousinvestigators in transplanting stem cells into hair follicles that areable to mature into functioning hair producing matrix cells. In oneexample, Hantash et al have taught of method of restoring hair by firstirradiating the skin to create elongated voids in the skin. Once thevoids created, stem cells such as hair follicle stem cells can betransplanted into the voids (U.S. Pat. App. Nos. 2007/0212335 and2012/0238941). They teach that stem cells can be harvested from avariety of sources including adult hair follicles. In other relatedworks, Cambier al taught a method to immortalize adult stem cellsobtained from harvesting stem cell types of interest from the tissue ororgan of interest for the treatment of human diseases (U.S. Pat. App.No. 2007/0116691). These immortalized stem cells can be used as a longterm source for transplantation.

In a very recent article by Toyoshima et al (Nat. Commun. 2012, 3, 784),the authors demonstrated fully functional hair regeneration in a nudemouse model using bioengineered hair follicle germ transplantedintracutaneously. The bioengineered hair follicle germ was abioengineered vibrissa follicle germ reconstituted with adultfollicle-derived cells. This approach is very promising buttransplantation requires invasive surgery to introduce the bioengineeredhair follicles. It was not yet determined if these engineered folliclesremain capable of producing hair in the long term. All of these studieswill need to reproduced in humans to completely understand if stem celltransplantation will work in human subjects.

A new alternative topical treatment for hair loss was reported using anFDA-approved glaucoma drug, bimatoprost, that is commercially availableto lengthen eyelashes may also be able to induce the growth of hair inthe human scalp. Clinical trials are underway to determine if thebimatoprost is effective beyond growing and thickening hair inandrogenetic alopecia where the hair follicle is deficient in progenitormatrix cells.

In a Canadian Application, CA2008/002019, related to the presentinvention, applicants reported that GCSF subcutaneously injected intomice at a similar dose as that used in humans, where the mice wereshaved and treated with depilatory cream, showed an approximate 20%increase in hair growth as well as it was noted the hair had thickershafts. Other factors in conjunction with GCSF promoting hair growth andhair thickening in mice included Vascular Endothelial Growth Factor andEpidermal Growth Factor. In same invention, the applicants used a modelof temporary hair loss to study the effect of GCSF or other factors.Mice were injected with cyclophosphamide, a chemotherapy drug, toinhibit hair growth. It should be noted that a side effect of GCSFtreatment in chemotherapy patients for neutropenia is hair loss.Surprisely, the applicants used over 10 fold higher dose rate perkilogram than used in human therapies and their previous study in patentapplication. In both untreated and treated mice, hair grew however therate of hair growth in mice injected with the large dose of GCSF showeda two fold increase in hair growth compared to control. It should benoted that GCSF was given one day after injection with cyclophosphamide,suggesting that the effect would be protective. The hair growth incontrol mice underscores that the model does not to accurately reflectthe deficient stem cells in androgenetic alopecia.

In the present invention, it was surprisingly found that GCSF was ableto re-grow hair when administered in multiple consecutive courses ofGCSF. Further, the hair that was restored was retained upon lessfrequent, routine administration of GCSF administered. In addition, GCSFwas able to increase both fingernail cuticle growth and density.

Example 1 Reversal of Symptoms with Parkinson's Disease withGranulocyte-Colony Stimulating Factor

The patient was a 61 year-old male with a six year history ofParkinson's Disease. When first diagnosed two years prior, the patientwas started on 100 milligrams Amantidine twice daily and 5 milligramsSelegiline twice daily and on increasing doses of Permax, 25 microgramsto 250 micrograms three times daily.

A history taken immediately before starting the dosing ofGranulocyte-Colony Stimulating Factor (GCSF) was typical of adult onsetParkinson's disease. The patient had blood work performed 7 days beforetreatment which included CBC, ESR, urine analysis and chemistry screen.In summary, the male patient, approximately 67 kg body weight diagnosedwith Parkinson's Disease had history of modest hyperlipidemia which wascontrolled on medications. Physical exam was normal except for somecogwheeling especially on the right. Granulocyte-Colony StimulatingFactor was started at a daily dose of 330 micrograms (1.1 ml) throughinjection for 5 consecutive days. Patient experienced slight bone painon second and third days of therapy. About 3 weeks afterGranulocyte-Colony Stimulating Factor therapy was started, patientrealized that he could smell, something that he had not been able to dofor more than 5 years. This observation is consistent with improvementin olfactory nerve function. About a week later, the patient found thatthe orthostatic hypotension that had given him trouble for the last 2-3years and had required him to wear support stockings, had disappeared.In addition, the patient was able to stand up quickly from a sittingposition or walk up stairs without becoming severely light-headed. Thepatient's handwriting, previously totally unreadable, was greatlyimproved and was legible to others. It was observed by his wife that thepatient had an improved facial affect. After about two months, a plateauwas reached.

Surprisingly, it was found that Granulocyte-Colony Stimulating Factortreatment of a patient with Parkinson's disease was followed four weekslater by quantifiable evidence of efficacy utilizing symptoms thatindicated an effect on both the Central Nervous System and PeripheralNervous System (PNS). The patient improved in several CNS symptoms thatincluded resting tremor, rigidity, postural instability, andmicrographia. Four accepted PNS symptoms of Parkinson's disease, theloss of sense of smell, orthostatic hypotension and urinary andgastrointestinal incontinence disappeared during the third and fourthweeks following the start of therapy. The disappearance of orthostatichypotension was not complete but was greatly reduced. Unexpectedly, GCSFcaused reversal of one or more symptoms of Parkinson's Disease upontreatment with the first dose. The reversed symptoms includedorthostatic hypotension, resting tremor, rigidity, postural instability,handwriting and urinary and gastrointestinal incontinence.

Example 2 Long Term Treatment of Parkinson's Disease UsingGranulocyte-Colony Stimulating Factor

The long term treatment of Parkinson's Disease by GCSF was followedusing a standardized system approved by neurologists, the UnifiedParkinson's Disease Scale (UPDS), to more completely access the efficacyof treatment. UPDS utilizes over 40 distinct clinical evaluations thatare predominantly based mostly on CNS symptoms. Analysis was performedapproximately 2 to 4 weeks after injection of the first dose. To morefully understand the effect of the GCSF, the data was analyzed todetermine whether a correlation existed between dose of GCSF andreversal of symptom score. The patient received injection of the samedose of GCSF every six to eight weeks and was periodically evaluated byUPDS criteria. In FIG. 1, the score of UPDS was plotted over the courseof two and one-half years. The correlation coefficient was −0.93suggesting the reversal was progressive upon GCSF treatment and wasstatistically significant (P=0.001). The final score obtained by thepatient approached the score that a non-patient might receive.

Because UPDS evaluates mostly CNS symptoms of Parkinson's Disease, fourindicators of PNS symptoms were also evaluated included urinary andgastrointestinal incontinence, orthostatic hypotension and loss ofsmell. The induction of orthostatic hypotension was evaluated by takingblood pressure before and after going from a lying down position tositting and then standing position and each time taking blood pressurewith an automatic apparatus. A drop of 50 mm was associated withdeveloping lightheadedness and orthostatic hypotension. All fourindicators were reversed relative to the state prior to GCSF treatment.In fact, the patient was able to exercise without fainting or having torest (orthostatic hypotension), enjoyed foods and wine (smell), resolvedurinary and gastrointestinal incontinence, and gained sense of smellresulting in a higher quality of life.

Example 3 Reduction of Blood Glucose Levels in Type 2 DiabeticsAdministered with Granulocyte-Colony Stimulating Factor

Other benefits were observed upon treatment with Granulocyte-ColonyStimulating Factor in the same patient as described in Example 1. Thepatient exhibited a two year history of slightly elevated blood glucosedeterminations (average fasting blood sugar ranging up to 115 mg/Dl).Patient had a slightly elevated hemoglobin A1C (6.5 versus normal of<6.0. He was diagnosed as Type 2 Diabetes and was recommended to aDiabetics clinic for classes on diet and exercise for dealing with thisdisease. Although dieting and exercise provided some reducing it was notsolely sufficient in controlling blood glucose levels below the 110mg/dl.

After treatment with Granulocyte-Colony Stimulating Factor it wasobserved that the blood sugar averaged 104 mg/Dl for 15 specimens beforetreatment and decreased to 92 mg/Dl during the period starting threeweeks after the start of drug (and at the same time that Parkinson'ssymptoms disappeared. The difference was highly significant (P=0.001).

Example 4 Long Term Controlled Reduction of Blood Glucose Levels in Type2 Diabetics Administered with Granulocyte-Colony Stimulating Factor

To examine the long term effect of Granulocyte-Colony Stimulating Factoron a patient with adult onset Type 2 diabetes, the patient in Example 2was monitored for approximately two and one-half years for fasting bloodglucose almost every morning. Starting with the first day of therapywith GCSF, every 5 day period was averaged and the mean used to compareblood glucose levels. FIG. 3 shows the analyzed data from blood sugardeterminations around six courses of GCSF. Each time after the GCSF isadministered the blood sugar dropped well within the normal range afterabout twenty to thirty days after the start of therapy. In one case drugwas not administered until late in the period of follow-up and then thenadir was reached about twenty-five days after the start of that course.All curves reached a nadir ranging between 20 and 30 days after thestart of therapy. Most blood glucose determinations remained below 110mg/Dl for about 2 months after treatment. It was unexpected that a drug,and more surprising GCSF, is able to reduce blood glucose for extendedperiods beyond one day, even more surprising one week and mostsurprising one month. The patient was also able to ingest limitedamounts of Dove® bars without any problems. GCSF provides a consistentand more desirable approach to regulate blood glucose levels in Type 2diabetics with less potential side effects of multiple mechanismsemployed to control Type 2 diabetes beyond other drugs.

Example 5 GCSF Supplemental Production of Stem Cells in a Close Donor orSelf to Improve Control in the Reduction of Blood Glucose Levels in Type2 Diabetics

An individual was treated with stem cells obtained from self or a closerelative as a way to increase the effect of GCSF in controlling on bloodglucose levels. In the later case, an identical twin was examined forMHC profile and found to be a match. To produce a supplemental amount ofstem cells in the identical twin, the twin was administeredGranulocyte-Colony Stimulating Factor a daily dose of 330 microgramsthrough injection for 5 consecutive days. Patient experienced slightbone pain on second and third days of therapy. After 7-10 days, theblood of the patient was subjected to leukaperesis to collect peripheralblood stem cells from the mobilized peripheral blood progenitors.Preferably, CD34+ stem cells are isolated from peripheral blood stemcells and HLA matched donors.

The stem cells obtained from a Neupogen-treated twin were injected intothe patient with Type 2 diabetes that was previously treated with GCSF.After 1-2 weeks, the patient was administered a course of GCSF; 330micrograms (1.1 ml) through injection for 5 consecutive days. It wasfound that the level of blood glucose decreased significantly from theprior treatment and remained lower for a longer period that the priortreatment.

The exogenous treatment using isolated stem cells of the Type 2 diabeticpatient was also repeated with the patient's own stem cells, however theincrease was less than that observed with treatment of stein obtainedfrom the identical twin. Preferably, self-produced stem are harvest fromthe individual when the individual is a young adult between the ages of16 to 35 years of age. The optimal ages are between 18 and 30 years ofage. The harvested stem cells are subjected to preservation methods,such as cryopreservation until needed.

Example 6 GCSF Acceleration of Hair Growth and Fingernail Growth andDensity

Yet other benefits of the Granulocyte-Colony Stimulating Factortreatment observed was the re-growth of hair in a human patient withandrogenic alopecia.

To re-grow of hair in the balding scalp, the patient was treated with anincreased frequency of the courses of Neupogen administered. Prior totreatment, the patient had received courses of GCSF as described inExample 2 and Example 4, however no significant re-growth of hair wasobserved. Prior to the increased frequency of treatment, the patientexhibited moderate hair loss and thinning, corresponding to a rating of5 on Hamilton-Norwood Scale for hair loss, ranging from 0 for no hairloss to 7 being complete hair scale loss. Neupogen (480 ug; 5-6 ug/kgbody weight) was administered for each day for four days followed by aperiod of no treatment for four days. This regimen was repeated for atotal of six times consecutively. After the patient continued on theirnormal periodic treatment of GCSF, being administered with GCSF coursesevery 6-18 weeks. Surprisingly, about 4-6 months after GCSF treatment,the substantial growth of new hair, or re-growth of hair, on thesubject's scalp resulted in a improvement of hair growth to 2 on theHamilton-Norwood Scale. The hair was similar in texture and distributionto early stages of male pattern baldness in the individual. In dramaticcomparison to the patient's identical twin, as shown in FIG. 5, theuntreated twin brother had a rating of 6 on the Hamilton-Norwood Scale.Both individuals had a similar amount of hair loss prior toadministration of GCSF to the treated patient.

After the increased courses were completed, the administration ofNeuprogen returned to a treatment of every 6-18 weeks. It was observedthe hair grew much more quickly in the months following administrationof the increased courses. The rate of hair growth and density has beenmaintained the same growth since the patient treatment has returned toroutine courses during the past 2 years. These findings showed that oncethe amount of desired hair growth is obtained, the lower frequency rateis suitable to retain hair growth.

For consecutive courses, GCSF is administered for about 3 to 6 days andthe treatment is repeated about every 3 to 8 days for two or moreconsecutive courses, preferably 3 to 6 courses. The administration ofeffective consecutive courses and periodic courses of GCSF isadministered at a daily dose rate of 0.1ug to 100 ug, preferably 0.1 to10 ug per kg, more preferably 0.1 to 5 ug per kg and most preferably 0.1to 2 ug per kg body weight.

Other factors can be used to supplement hair growth and density withGCSF administration. For example, based on bimaprost findingsstimulating hair growth and thickening, bimaprost, and like compoundsworking on related receptors in the follicle area, may be effective toincrease hair volume once the damaged progenitor stem cells have beenreplenished. Other factors that may enhance include Vascular EndothelialGrowth Factor and Epidermal Growth Factor reported earlier to stimulatehair growth. GCSF treatment would also be effective as a companiontreatment for stem cell transplantation either prior to, after or both,including pre-conditioning of the stem cells with GCSF prior totransplantation,

This is the first report where GCSF was demonstrated to re-store hairgrowth in a human patient with androgenic alopecia. The results showedthat GCSF was able to restore hair matrix cells functionality to growthhair and further treatments could obtain additional hair growth. Theconsecutive course regimen of GCSF not only mobilizes progenitor stemcells but also facilitates their maturation into hair producing matrixcells. It is surprising that many references have suggested that hairloss may occur with GCSF treatment, however this may be complicated bythe fact that GCSF in limited courses in chemotherapy.

Because the hair matrix cells are similar to fingernail matrix cells, itwas also observed that the cuticle density and growth improveddramatically during the same period. Prior to treatment, the fingernailsof patient were very flexible and had a slow growth rate as well as thenails were often chipped. In the same subject treated with the multiplecourses of GCSF administration described for hair re-growth, it wasfound that the fingernails had become much denser and grew at a fasterrate similar to that observed for their hair. Identical effectiveconsecutive courses and periodic courses of GCSF for hair would used befor cuticle, fingernail and toe nail GCSF treatments.

OTHER EMBODIMENTS

The description of the specific embodiments of the invention ispresented for the purposes of illustration. It is not intended to beexhaustive nor to limit the scope of the invention to the specific formsdescribed herein. Although the invention has been described withreference to several embodiments, it will be understood by one ofordinary skill in the art that various modifications can be made withoutdeparting from the spirit and the scope of the invention, as set forthin the claims. All patents, patent applications, and publicationsreferenced herein are hereby incorporated by reference. Otherembodiments are within the claims.

1-8. (canceled)
 9. A method for increasing cuticle growth and density by administering effective consecutive courses of Granulocyte-Colony Stimulating Factor, wherein said Granulocyte-Colony Stimulating Factor is selected from a group consisting of Granulocyte-Colony Stimulating Factor, its derivative, functionally similar biological or chemical compound or combination thereof.
 10. The method of claim 9, wherein said effective consecutive courses of Granulocyte-Colony Stimulating Factor for increasing cuticle growth and density are two or more consecutive courses repeated about every 3 to 8 days, wherein said consecutive course administers a daily dose of Granulocyte-Colony Stimulating Factor for about 3 to 6 days.
 11. The method of claim 10, wherein said daily dose of Granulocyte-Colony Stimulating Factor administers a Granulocyte-Colony Stimulating Factor dose of about 0.1 micrograms to 100 micrograms per kg body weight per day of Granulocyte-Colony Stimulating Factor on each day.
 12. The method of claim 9, wherein said cuticle is a fingernail or a toe nail.
 13. The method of claim 10, wherein said Granulocyte-Colony Stimulating Factor daily dose is administered selected from a group consisting of subcutaneous injection, transdermal patch, intravenously, and orally.
 14. (canceled)
 15. The method of claim 1, further comprising maintaining cuticle growth and density by a periodic effective course of Granulocyte-Colony Stimulating Factor after said consecutive courses are completed, wherein said treatment maintains the cuticle growth and density.
 16. The method of claim 15, wherein said periodic effective course of Granulocyte-Colony Stimulating Factor to maintain cuticle growth and density is a course of Granulocyte-Colony Stimulating Factor administered daily for about 3 to 6 days and said course is repeated every 2 to 18 weeks, wherein daily dose of Granulocyte-Colony Stimulating Factor is a dose of about 0.1 micrograms to 100 micrograms per kg body weight per day of Granulocyte-Colony Stimulating Factor on each day.
 17. The method of claim 15, wherein said administered is selected from a group consisting of subcutaneous injection, transdermal patch, intravenously, and orally.
 18. (canceled)
 19. A method for maintain cuticle growth and density by administering periodic effective courses of Granulocyte-Colony Stimulating Factor, wherein said Granulocyte-Colony Stimulating Factor is selected from a group consisting of Granulocyte-Colony Stimulating Factor, its derivative, functionally similar biological or chemical compound or combination thereof. 